Type IV pili are important bacterial virulence factors that mediate attachment to mammalian host cells and elicit downstream signals. When adhered to abiotic surfaces, the human pathogen Neisseria gonorrhoeae generates force by retracting these polymeric cell appendages. We recently found that single pili generate stalling forces that exceed 100 pN, but it is unclear whether bacteria generate force once they adhere to their human host cells. Here, we report that pili retract very actively during infection of human epithelial cells. The retraction velocity is bimodal and the high velocity mode persisted at higher forces in contrast to an abiotic environment. Bacteria generate considerable force during infection, but the maximum force is reduced from 120+/-40 pN on abiotic surfaces to 70+/-20 pN on epithelial cells, most likely due to elastic effects. Velocity and maximum force of pilus retraction are largely independent of the infection period within 1 h and 24 h post-infection. Thus, the force generated by type IV pili during infection is high enough to induce cytoskeletal rearrangements in the host cell.